(1) Goals of project: - To develop sensitive assay for monitoring HIV-1 envelope-mediated cell fusion. - To identify surface receptors(in addition to the CD4 receptors) involved in HIV-1 fusion and cell entry. - To apply the knowledge gained towards development of agents capable of blocking infection by cell-free or cell-associated HIV-1 (2) Experimental approach: - We have developed a sensitive assay to examine the early stages of HIV-1 env mediated cell membrane fusion which is based on redistribution of fluorescent dyes between HIV-1 env-expressing cells and CD4-positive adjacent cells, monitored by fluorescence video microscopy. (3) Major Findings: - Our study demonstrated that HIV-1 mediated cell fusion and syncytia formation followed different kinetics. Furthermore, fusion occurred under conditions (i.e. cell ratios) which did not favor syncytia formation (similar to the in vivo situation). - This assay was used to measuere the kinetics of fusion between HIV-env expressing cells and truncated CD4 molecules or chiemic CD4.CD8 molecules.- We have identified a second membrane compenent which is required for HIV-1 fusion and can be down-modulated by the PKC activator, phorbol ester (PMA). It was also found that gp120 interaction with CD4 induces its association with the second components forming a tri- molecular complex (gp120-X-CD4) which may stabilize the fusion process, and aggregates in clathrin coated pits. Biochemical assays are underway to define component X (secondary receptor for HIV entry?)